Choriocarcinoma Cell Line BeWo Research Articles

Gestational Diabetes Mellitus-Induced Inflammation in the Placenta via IL-1β and Toll-like Receptor Pathways.

Gestational diabetes mellitus is characterised by an insufficient insulin response to hyperglycaemia and the development of insulin resistance. This state has adverse effects on the health outcomes of the mother and child. Existing hyperglycaemia triggers a state of inflammation that involves several tissues, including the placenta. In this study, we analysed the putative pathomechanism of GDM, with special emphasis on the role of chronic, sterile, pro-inflammatory pathways. The expression and regulation of the elements of IL-1β and Toll-like receptor (TLR) pathways in GDM maternal blood plasma, healthy placental explants and a choriocarcinoma cell line (BeWo cell line) stimulated with pro-inflammatory factors was evaluated. Our results indicate elevated expression of the IL-1β and TLR pathways in GDM patients. After stimulation with IL-1β or LPS, the placental explants and BeWo cell line showed increased production of pro-inflammatory IL-6, TNFa and IL-1β together with increased expression of the elements of the signalling pathways. The application of selected inhibitors of NF-ĸB, MAPK and recombinant interleukin 1 receptor antagonist (IL1RA) proved the key involvement of the IL-1β pathway and TLRs in the pathogenesis of GDM. Our results show the possible existence of loops of autocrine stimulation and a possible inflammatory pathomechanism in placentas affected by GDM.

Equol exerts anti-tumor effects on choriocarcinoma cells by promoting TRIM21-mediated ubiquitination of ANXA2

Choriocarcinoma is a malignant cancer that belongs to gestational trophoblastic neoplasia (GTN). Herein, serum metabolomic analysis was performed on 29 GTN patients and 30 healthy individuals to characterize the metabolic variations during GTN progression. Ultimately 24 differential metabolites (DMs) were identified, of which, Equol was down-regulated in GTN patients, whose VIP score is the 3rd highest among the 24 DMs. As an intestinal metabolite of daidzein, the anticancer potential of Equol has been demonstrated in multiple cancers, but not choriocarcinoma. Hence, human choriocarcinoma cell lines JEG-3 and Bewo were used and JEG-3-derived subcutaneous xenograft models were developed to assess the effect of Equol on choriocarcinoma. The results suggested that Equol treatment effectively suppressed choriocarcinoma cell proliferation, induced cell apoptosis, and reduced tumorigenesis. Label-free quantitative proteomics showed that 136 proteins were significantly affected by Equol and 20 proteins were enriched in Gene Ontology terms linked to protein degradation. Tripartite motif containing 21 (TRIM21), a E3 ubiquitin ligase, was up-regulated by Equol. Equol-induced effects on choriocarcinoma cells could be reversed by TRIM21 inhibition. Annexin A2 (ANXA2) interacted with TRIM21 and its ubiquitination was modulated by TRIM21. We found that TRIM21 was responsible for proteasome-mediated degradation of ANXA2 induced by Equol, and the inhibitory effects of Equol on the malignant behaviors of choriocarcinoma cells were realized by TRIM21-mediated down-regulation of ANXA2. Moreover, β-catenin activation was inhibited by Equol, which also depended on TRIM21-mediated down-regulation of ANXA2. Taken together, Equol may be a novel candidate for the treatment for choriocarcinoma.Graphical abstract

Downregulation of SPARC Expression Enhances the Fusion of BeWo Choriocarcinoma Cells.

Syncytiotrophoblasts, which are formed by the fusion of villous cytotrophoblasts, play an essential role in maintaining a successful pregnancy. Secreted protein acidic and rich in cysteine (SPARC) is a non-structural Ca2+-binding extracellular matrix glycoprotein involved in tissue remodeling and cell proliferation, differentiation, and migration. Previous studies have revealed that SPARC is expressed in villous and extravillous cytotrophoblasts in the first trimester and that RNA interference targeted at SPARC significantly inhibited invasion of human extravillous trophoblast HTR8/SVneo cells. However, the involvement of SPARC in cytotrophoblast fusion remains unknown. This study aimed to investigate the role of SPARC in cytotrophoblast fusion, using the BeWo choriocarcinoma cell line as a model of villous cytotrophoblasts. Immunohistochemical analysis was conducted to assess SPARC expression in normal human placentas using placental tissues obtained during the first and third trimesters of pregnancy. We investigated the effects of SPARC knockdown on trophoblast differentiation markers and cell fusion in BeWo cells using small interfering RNA. Immunohistochemical analysis revealed that SPARC expression was high in the early gestational chorionic villi and low in the late gestational chorionic villi. SPARC knockdown increased the expressions of human chorionic gonadotropin and Ovo-like transcriptional repressor 1; however, glial cells missing transcription factor 1, syncytin-1, and syncytin-2 showed no significant changes. The assessment revealed that SPARC knockdown significantly enhanced cell fusion compared to the non-silencing control. Our data suggest that SPARC plays a vital role in regulating trophoblast fusion and differentiation during placental development.

Sirtuin 1 is a potential therapeutic candidate gene for fetal growth restriction via insulin-like 4

Objective Insufficient placental development causes various obstetric complications, including fetal growth restriction (FGR). The Sirtuin 1 (SIRT1) and insulin-like 4 (INSL4) protein-coding genes have been demonstrated to play an important role in placental development. However, no treatment for FGR is available due to placental dysfunction. Therefore, this study aimed to examine the potential of the SIRT1–INSL4 axis as a treatment candidate for FGR caused by insufficient placental development. Methods Twenty patients were enrolled, including 10 with FGR and 10 full-term controls. FGR and control placental samples were collected. Quantitative real-time polymerase chain reaction, immunohistochemical analysis, and western blotting were used to analyze INSL4 and SIRT1 expression. An in-vitro loss-of-function approach with the human choriocarcinoma cell line BeWo was applied for functional analyses of SIRT1 in placental development. BeWo cells were differentiated into syncytiotrophoblasts by silencing SIRT1 using small interfering RNA. SIRT1 activator was added during differentiation of SIRT1-knockdown BeWo cells into syncytiotrophoblasts. Results The FGR samples had lower INSL4 and SIRT1 mRNA and protein expression levels than the control samples. Immunohistochemistry showed that both SIRT1 and INSL4 were expressed mainly in syncytiotrophoblasts. In-vitro analyses showed that SIRT1 knockdown decreased INSL4 expression; however, SIRT1 activator restored SIRT1 expression in SIRT1-silenced BeWo cells. Conclusions SIRT1 and INSL4 are downregulated in the placenta of FGR, and INSL4 is regulated by SIRT1. These findings indicate that the SIRT1–INSL4 axis may be a potential therapeutic target for FGR.

All-Trans Retinoic Acid Reduces Matrix Metalloproteinase-2 and Increases E-Cadherin Levels in BeWo Choriocarcinoma Cells

Background: Choriocarcinoma is a malignant trophoblastic tumor that can degrade the uterine basement membrane to facilitate local and distant metastasis. Matrix metalloproteinase-2 (MMP-2) is a vital proteinase produced by trophoblasts and can cleave type IV collagen in the basement membrane of the uterine epithelium. In addition, E-cadherin controls cell adhesion, which is associated with tumorprogression, and loss of E-cadherin function is associated with metastasis. Vitamin A analogues, including all-trans retinoic acid (ATRA), exhibit anticancer properties, including metastasis inhibition.This study aimed to investigate the ability of ATRA to prevent trophoblast invasion and metastasis in choriocarcinoma by assessing MMP-2 and E-cadherin activity. Methods: This study administered various doses of ATRA to the BeWo (American Type Culture Collection CCL-98) choriocarcinoma cell line and assessed MMP-2 and E-cadherin activity through flow cytometry. The research was performed at the Biomedical Laboratory of the Faculty of Medicine of Universitas Brawijaya Malang, East Java, Indonesia. Results: BeWo cells were exposed to 0, 50, 100, 200, 400, or 800 μg/mL ATRA. MMP-2 was downregulated after ATRA treatment. The greatest reduction in MMP-2 level was observed after 200 μg/mL ATRA treatment, but this result was not statistically significant (P = 0.550). However, ATRA treatment significantly reduced the number of BeWo cells with low E-cadherin levels (P = 0.012). Conclusion: ATRA downregulates MMP-2 and upregulates E-cadherin. Further in vitro and animal studies are required to evaluate the ability of ATRA to inhibit metastasis. Bangladesh Journal of Medical Science Vol. 22 No. 02 April’23 Page : 336-340

Stromal cell senescence contributes to impaired endometrial decidualization and defective interaction with trophoblast cells.

What are the consequences of endometrial stromal cell (EnSC) senescence for endometrial function? Senescence of EnSC contributes to impaired endometrial decidualization and impaired interaction with trophoblast cells but application of senomorphics diminishes the adverse effects of senescent EnSC on decidualization and implantation. A prolonged and highly disordered pro-inflammatory secretory profile of EnSC, which resembles the senescence-associated secretory phenotype, is associated with implantation failure. Furthermore, it has been suggested that implantation failure may be associated with increased EnSC senescence during the proliferative phase of the menstrual cycle. Primary EnSC cell cultures were isolated from endometrial biopsies taken from four patients without any endometrial complications planning to undergo IVF. EnSC senescence was induced by oxidative stress (1 h exposure to 200 µM H2O2) followed by 14 days culture but some results were confirmed in a replicative senescence model (after 25 passages). The decidual reaction was evaluated with routine methods and a genetic tool previously designed by us that estimates integral decidual response by fluorescence of a reporter protein. Time-course RNA-sequencing of control and senescent EnSC before and during decidualization was performed using four replicates for each state. To extend our findings, we applied several publicly available datasets. To model implantation in vitro, the choriocarcinoma cell line BeWo b30 was used. To reduce the senescent phenotype of EnSC, two classical senomorphics were applied-rapamycin and metformin. EnSC cultures were used to investigate the effects of senescence on decidualization and on an in vitro implantation model using spheroids derived from BeWo cells. Co-culture models (2D and 3D) were used to explore the effect of senescent cells on neighbouring control cells. The following methods were used to assess cell function, RNA-sequencing, bioinformatic analysis, CRISPR/Cas9 genome editing, FACS, western blotting, RT-PCR, immunofluorescence, molecular cloning, lentiviral transduction and ELISA. Premature senescence of EnSC could be a cause of impaired decidualization. Hormone-induced decidual transformation of EnSC cultures was negatively affected by senescence. Bioinformatics revealed crucial disturbances in the decidual reaction of senescent EnSC which could affect embryo invasion, alter the 'meta-signature' of human endometrial receptivity, disturb the emergence of mature and senescent decidual cells subpopulations, impair ligand-receptor interaction with trophoblasts and modify the architecture of extracellular matrix. These predictions were functionally validated using an in vitro implantation model. Moreover, we observed that senescent EnSC, likely via the altered secretome, caused 'bystander' quenching of the decidual reaction in adjacent cells, reinforcing dysfunction of the stromal compartment. Application of senomorphics that reduced the senescence phenotype diminished adverse effects of senescent EnSC on decidualization and implantation. The data used in this study are available in the GEO database (GEO identifier GSE160702). The present study was based on in vitro cell cultures derived from only four women. Further studies involving patients with impaired implantation are needed to confirm our findings. The presence of senescent EnSC within the stromal compartment of the endometrium may be a risk-factor for the failure of embryo implantation. Application of senomorphics during the proliferative phase of the menstrual cycle is a promising strategy to alleviate negative effects of senescent EnSC and to improve embryo implantation rates. This study was funded by the Russian Science Foundation (# 19-74-10038). The authors do not have any competing interests to declare.

Syncytialization of BeWo trophoblasts induces changes in angiogenic signaling and response to hypoxia

During pregnancy, the placenta produces significant amounts of pro and anti‐angiogenic factors, notably VEGF, PlGF, and sFlt‐1. The exact cell population(s) most responsible for this production is not definitively known. In the event of placental insufficiency, placental ischemia can cause alterations in the production of these factors, favoring the anti‐angiogenic sFlt‐1 protein. This mechanism is believed to be a major driver of preeclampsia. Within the placenta, the maternal/fetal interface is defined by a layer of specialized syncytiotrophoblasts (ST) which line the maternal side of the underlying fetal chorionic vessels. These large multinucleated cells define the selectivity of nutrient/waste exchange from the maternal to the fetal circulation. In studying trophoblast function, immortalized trophoblast‐derived cell lines are commonly utilized, such as the BeWo choriocarcinoma cell line. These cells however, do not spontaneously syncytialize, and therefore do not likely reflect in vivo ST function. We hypothesized that inducing syncytialization of BeWo cells would alter their production of angiogenic factors in vitro. Syncytialization of BeWo cells was induced with forskolin treatment for 48 hours. At 48 hours, cellular morphology shifted from dense cobblestone patterning into majority large, multinucleated cells. Syncytialization caused ~5 fold increase in both VEGF and PlGF mRNA (p<0.0001). There were significant (p<0.0001) increases in the levels of sFlt‐1 splice variants 2 (~5 fold), 3 (~50 fold), and 4 (~3fold). As a result, there were significant (p,<0.005) ~2 fold increases in secreted VEGF and PlGF protein, while sFlt‐1 went from not detectable to 53 pg/ml as measured by ELISA. Perhaps most interestingly, when cultured in oxygen concentrations mimicking healthy and ischemic placentas (8% and 1% respectively), there was differential regulation of these factors at the transcriptional level. There was no change in VEGF, while sFlt V2 and V3 were significantly upregulated (2‐fold each, p<0.0005), while PlGF was significantly down regulated by 40% (p<0.0001). Taken together, these data suggest that syncytialization significantly alters expression of both pro and anti‐angiogenic factors in vitro. Future studies should take syncytialization state into account when extrapolating from cell culture to in vivo placental function.

Cadmium inhibits forskolin-induced differentiation of human placental BeWo cells.

Cadmium (Cd) is an environmental pollutant. Blood Cd levels in pregnant women have been associated with premature births, infant birth size, placenta previa, and placenta accreta. There have been concerns on the reproductive developmental toxicity of Cd. The choriocarcinoma cell line BeWo, a cellular in vitro model for studying syncytial fusion, has been widely used to study the reproductive and developmental toxic effects of pollutants. Here, we examine the inhibitory effect of Cd against forskolin (FSK)-induced BeWo differentiation. Results showed that Cd exposure inhibited the FSK-induced expression of syncytiotrophoblast-related genes LGALS13, ERVFRD1, SDC1, and CGB3. Inhibition of LGALS13 expression was due to the inhibition of the PKA pathway, whereas the inhibition of the other three genes could be due to the inhibition of the other pathways. These findings could help clarify the reproductive and developmental toxicity of Cd.

Insulin-like growth factor 1 signaling in the placenta requires endothelial nitric oxide synthase to support trophoblast function and normal fetal growth.

Currently, there is no effective treatment for placental dysfunction in utero. In a ligated mouse model of fetal growth restriction (FGR), nanoparticle-mediated human insulin-like 1 growth factor (hIGF1) gene delivery (NP-Plac1-hIGF1) increased hIGF1 expression and maintained fetal growth. However, whether it can restore fetal growth remains to be determined. Using the endothelial nitric oxide synthase knockout (eNOS-/-) mouse model, a genetic model of FGR, we found that despite inducing expression of hIGF1 in the placentas treated with NP-Plac1-hIGF1 (P = 0.0425), FGR did not resolve. This was associated with no change to the number of fetal capillaries in the placental labyrinth; an outcome which was increased with NP-Plac1-hIGF1 treatment in the ligated mouse model, despite increased expression of angiopoietin 1 (P = 0.05), and suggested IGF1 signaling in the placenta requires eNOS to modulate placenta angiogenesis. To further assess this hypothesis, BeWo choriocarcinoma cell line and human placental explant cultures were treated with NP-Plac1-hIGF1, oxidative stress was induced with hydrogen peroxide (H2O2), and NOS activity was inhibited using the inhibitor NG-monomethyl-l-arginine (l-NMMA). In both BeWo cells and explants, the protective effect of NP-Plac1-hIGF1 treatment against H2O2-induced cell death/lactate dehydrogenase release was prevented by eNOS inhibition (P = 0.003 and P < 0.0001, respectively). This was associated with an increase in mRNA expression of oxidative stress markers hypoxia inducing factor 1α (HIF1α; P < 0.0001) and ADAM10 (P = 0.0002) in the NP-Plac1-hIGF1 + H2O2 + l-NMMA-treated BeWo cells. These findings show for the first time the requirement of eNOS/NOS in IGF1 signaling in placenta cells that may have implications for placental angiogenesis and fetal growth.

1112HAND1 knockdown disrupts trophoblast but not placental endothelial global gene expression

We previously reported disrupted placental structure, and vascular changes in placentas from fetuses with Congenital Heart Defects. Signaling between trophoblast and villous endothelium is proposed for proper placental development and function. We hypothesize that disruption of genes associated with CHD within the trophoblast impacts fetal endothelium and results in dysregulated placental vasculature in CHD fetuses. A model of human cytotrophoblast, BeWo choriocarcinoma cells and isolated human placental microvascular endothelial cells (HPMVEC) were treated for 48 hrs with HAND1 siRNA/lipofectamine. Additionally, control HPMVECs were exposed to conditioned BeWo media (+/- HAND1) for 48 hrs. Cells were harvested and total RNA isolated. RNA was sequenced on an Illumina NextSeq 550 platform (single end reads 85bp, read depth 25M). The Galaxy Bioinformatic Platform was used for quality control, alignment (Bowtie2) and generation of read counts (Feature Counts). Normalization and differential gene expression analyses were conducted using general linear modeling in edgeR package from Bioconductor. Significance was determined using a fold change cut off of >1.5 and raw p<0.05. Overrepresentation analysis was performed using Panther DB for Reactome pathways and GO terms Direct HAND1 knockdown in the BeWo choriocarcinoma cell line caused downregulation of 632 genes, and upregulation of 102 genes. Overrepresentation analysis identified disruption to pathways including cell differentiation, neurogenesis, localization, and cell projection organization. In contrast, direct HAND1 knockdown in HPMVECs resulted in minimal disruption to global gene expression with differential expression in just 18 genes. Indirect HAND1 disruption did not alter HPMVEC gene expression. Disruption to HAND1 expression significantly alters gene expression profile in trophoblast but not HPMVEC, either directly or indirectly. Disruption to the structure and vasculature of the placenta in cases of CHD is likely primarily due to abnormal trophoblast development.

315 Localization and differential expression of HLA-G in term human fetal membranes

Pregnancy requires immunologic tolerance to support the semiallogenic fetus. HLA-G, a non-classical type I MHC molecule, plays an immunomodulatory role and its expression is reported in placental extravillous trophoblasts. This study localized HLA-G in human fetal membrane cell layers and determined its differential expression in term not in labor (TNIL) or term labor (TL) samples as an indicator of immunologic changes during parturition. Fetal membranes were collected from TNIL (N=6) cesarean and TL (N=6) vaginal deliveries. To localize HLA-G, paraffin embedded sections were immunostained with antibody specific to HLA-G. To determine cell layer and cell type specific HLA-G expression, membranes were divided into amnion and chorion (TNIL [N=4], TL [N=6]) or dispersed and grown as primary cells (amnion epithelial cells [AEC], amnion mesenchymal cells [AMC], chorion trophoblast cells [CTC], decidual cells [DEC]). Protein extracts from layers or cells were analyzed by western blot to determine difference in HLA-G expression between TL and TNIL samples. BeWo (choriocarcinoma cell lines) were used as negative control. Regardless of labor status, amnion and chorion express HLA-G. Although not quantitative, staining intensity was higher in chorion (Fig 1). This was confirmed by western blot. Chorion and CTC had higher HLA-G expression than amnion and AEC; AMC of the membrane matrix were negative for HLA-G (Fig 2a). Quantitative differences were not observed between TNIL and TL in any of the cell types expressing HLA-G (Fig 2b). Decidual layer and BeWo cells were negative for HLA-G. This is the first report to localize HLA-G to the components of the fetal membrane and demonstrates that HLA-G is expressed predominantly in chorion and to a lesser extent in amnion. HLA-G in the membrane likely contributes to immune regulation in pregnancy. Lack of change at term suggests that expression is constitutive in the membrane and differential expression is not a prerequisite for parturition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Identification and Functional Assessment of the First Placental Adhesin of Treponema pallidum That May Play Critical Role in Congenital Syphilis.

Syphilis is a global, re-emerging sexually transmitted infection and congenital syphilis remains a major cause of adverse pregnancy outcomes due to bacterial infection in developing nations with a high rate of fetus loss. The molecular mechanisms involved in pathogenesis of the causative agent, Treponema pallidum subsp. pallidum remain poorly understood due to the difficulties of working with this pathogen, including the inability to grow it in pure culture. To reduce the spread of syphilis, we must first increase our knowledge of the virulence factors of T. pallidum and their contribution to syphilis manifestations. Tp0954 was predicted to be a surface lipoprotein of T. pallidum. Therefore, we experimentally demonstrated that Tp0954 is indeed a surface protein and further investigated its role in mediating bacterial attachment to various mammalian host cells. We found that expression of Tp0954 in a poorly adherent, but physiologically related derivative strain of the Lyme disease causing spirochete Borrelia burgdorferi B314 strain promotes its binding to epithelial as well as non-epithelial cells including glioma and placental cell lines. We also found that Tp0954 expression facilitates binding of this strain to purified dermatan sulfate and heparin, and also that bacterial binding to mammalian cell lines is mediated by the presence of heparan sulfate and dermatan sulfate in the extracellular matrix of the specific cell lines. These results suggest that Tp0954 may be involved not only in initiating T. pallidum infection by colonizing skin epithelium, but it may also contribute to disseminated infection and colonization of distal tissues. Significantly, we found that Tp0954 promotes binding to the human placental choriocarcinoma BeWo cell line, which is of trophoblastic endocrine cell type, as well as human placental tissue sections, suggesting its role in placental colonization and possible contribution to transplacental transmission of T. pallidum. Altogether, these novel findings offer an important step toward unraveling syphilis pathogenesis, including placental colonization and T. pallidum vertical transmission from mother to fetus during pregnancy.

Factors Involved in miRNA Processing Change Its Expression Level during Imitation of Hypoxia in BeWo b30 Cells.

One of the main complications of pregnancy and causes of maternal and perinatal mortality is preeclampsia. The pathophysiology of preeclampsia is associated with the development of placenta and fetal hypoxia and secretion of a number of effective molecules. The human choriocarcinoma cell line BeWo b30 is often used as a model of the placental barrier. It was shown that oxyquinoline derivatives can mimic hypoxia by suppressing HIF-prolyl hydroxylases and the accumulation of HIF-1α. This effect also leads to a change in the expression of microRNAs and their target genes. However, with hypoxia in cells, not only the level of individual miRNAs but also the ratio of miRNA isoforms (isomiRs) can change, presumably due to inaccuracies in the work of the Drosha and Dicer enzymes. In this work, we showed a change in the expression of the factors involved in the maturation of miRNAs when simulating hypoxia in BeWo b30 cells with an oxyquinoline derivative, which may be one of the causes for the change in the ratio of miRNA isoforms.

Activated \u03b12-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α2-macroglobulin (α2M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α2M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α2M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α2M* on syncytialisation. We first demonstrated that α2M* induced trophoblastic cell fusion. This effect is dependent on α2M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.

OR14-07 Association Between Placental Glucose Uptake and Protein O-Glcnacylation and Birth Outcomes in Obese Non-Diabetic Mothers

Increased transport of nutrients such as glucose, across the placenta, has been linked to abnormal fetal growth and pregnancy complications in obese non-diabetic mothers (1); however, the underlying mechanisms are poorly understood. We hypothesized that in placenta, the metabolic and nutrient sensor O-GlcNAc transferase (OGT), highly sensitive to glucose flux through the hexosamine biosynthetic pathway (HBP), responds to maternal obesogenic environment by increasing O-GlcNAc post-translational modification of nucleocytoplasmic proteins in the placenta altering fetal growth trajectories. Tissue biopsies were isolated from placentas collected at term from 26 non-diabetic mothers alongside routine biochemistry and anthropometric data (maternal fasting glucose, glycated hemoglobin (HbA1c), early pregnancy body weight (BMI) and birth weight). OGT and glucose transporter 1 (GLUT1) protein expression as well as tissue levels of O-GlcNAcylation were determined by immunoblotting using specific antibodies. The BeWo choriocarcinoma cell line was also used as an in vitro model of trophoblast to test the effect of high glucose and GLUT1 silencing on the OGT activity by immunoblotting. Maternal BMI was positively correlated to birth weight centile (BWC) (p=0.0130, R2=0.231), maternal fasting glucose (p=0.0177, R2=0.221) and HbA1c levels (p= 0.0156, R2=0.229) as well as placental OGT protein expression (p=0.0294, R2=0.183). The latter was positively associated to the levels of protein O-GlcNAcylation (p=0.0023, R2=0.326). Interestingly, GLUT1 protein levels were positively correlated to BWC (p=0.0056, R2=0.279) and strongly correlated to protein O-GlcNAcylation (p<0.0001, R2=0.507), suggesting an increase in the placental flux of glucose. In agreement with findings in placenta biopsies, in BeWo cells total protein O-GlcNAcylation levels were altered by cell exposure to different glucose levels (5 mM vs 15 mM, p<0.01). This was prevented by downregulating OGT or GLUT1 expression (p<0.001) using gene silencing. In addition, OGT protein levels were negatively associated to AMP-activated protein kinase (AMPK) activation (p=0.0005, R2=0.402) in placenta biopsies identifying a novel cross-talk between two placental nutrient sensors, OGT and AMPK, previously shown in other tissues (2). Accordingly, the silencing of OGT promoted the activation of AMPK (p<0.01) and its downstream target acetyl-CoA carboxylase (ACC) (p<0.01) in BeWo cells, as demonstrated by increased phosphorylation of residues Thr172 and Ser79 for AMPK and ACC respectively. Such obesity-associated cross talk between metabolic and nutrient sensors might disrupt multiple cellular pathways involved in fetal development and growth.

Maternal dexamethasone exposure suppresses placental iron transport through GR‐mediated TFR1 pathway in rats

Iron is one of the essential trace elements for almost all living organisms, and of course it is especially important for pregnancy and fetal development. However, inappropriately low or high levels of iron during pregnancy are detrimental and contribute to serious and lasting effects on the fetus, such as fetal growth and development disorders, thereby increasing the risk of adulthood. Here we stress pregnant rats by drinking water with 10−7 mol/L dexamethasone, and then explore its effects on iron metabolism of placenta and fetal rats. The results showed that dexamethasone treatment inhibited placental development, down‐regulated the expression of placental iron transporter TFR1, and decreased iron levels in fetal rats. Furthermore, we use a human placental choriocarcinoma cell line (BeWo) as model to explore the pathway linking inhibit of TFR1 under Dex challenge, our results demonstrate that the decrease in TFR1 caused by dexamethasone is alleviated by the addition of dexamethasone inhibitor RU486. Taken together, our results show that dexamethasone downregulate TFR1 expression adjust iron metabolism via GR‐mediated.Support or Funding InformationThis work was supported by grants from the National Key Research and Development Program of China (2016YFD0500502), the National Natural Science Foundation of China (31872439), the Priority Academic Program Development of Jiangsu Higher Education Institutions and the Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control.

Anticardiolipin from Periodontitis Patients Impact Fetal Loss and Annexin V

Anticardiolipin antibodies, found at elevated serum concentrations in 15% to 20% of individuals with periodontitis, are associated with adverse pregnancy outcomes, thrombotic conditions, and accelerated atherosclerosis in autoimmune disease such as the antiphospholipid syndrome. Our previous studies demonstrated that antibodies raised in mice against Porphyromonas gingivalis caused fetal loss in a mouse pregnancy model due to anticardiolipin antibodies. Such antibodies are induced via molecular mimicry with the serum protein β2-glycoprotein 1 (β2GP1), the target antigen of anticardiolipin. Furthermore, human anticardiolipin IgG is associated with increased serum markers of vascular inflammation, and IgG purified from periodontitis subjects with elevated anticardiolipin stimulates inflammatory cytokine production by endothelial cells and a trophoblastic cell line. Activation of the trophoblastic cells by anticardiolipin occurs through Toll-like receptor 4. In the present study, we observed that IgG anticardiolipin from periodontitis subjects also causes fetal loss in mice. Displacement of the protective 2-dimensional lattice formed by annexin V on trophoblast surfaces by anticardiolipin, via its interaction with its target antigen β2GP1, leading to fibrin clot formation due to exposure of anionic phospholipids to plasma, is a plausible pathogenic mechanism explaining adverse obstetrical outcomes in antiphospholipid syndrome. Therefore, we assessed such interactions in periodontitis. We observed that anticardiolipin from periodontitis subjects competes for annexin V on an artificial phosphatidylserine monolayer, replicating a key activity of autoantibodies found in patients with antiphospholipid syndrome. In addition, we found that anticardiolipin from periodontitis subjects increases annexin V levels on the BeWo choriocarcinoma cell line, consistent with mobilization of annexin V to the cell surface to facilitate repair following membrane damage. The data indicate that sera and IgG from periodontitis subjects with elevated anticardiolipin levels may influence pregnancy outcomes due to interactions with annexin V.

Reactive oxygen species from mitochondria impacts trophoblast fusion and the production of endocrine hormones by syncytiotrophoblasts.

The placenta, a tissue that is metabolically active and rich in mitochondria, forms a critical interface between the mother and developing fetus. Oxidative stress within this tissue, derived from the dysregulation of reactive oxygen species (ROS), has been linked to a number of adverse fetal outcomes. While such outcomes have been associated with mitochondrial dysfunction, the causal role of mitochondrial dysfunction and mitochondrially generated ROS in altering the process of placentation remains unclear. In this study, mitochondrial complex I activity was attenuated using 10 nM rotenone to induce cellular oxidative stress by increasing mitochondrial ROS production in the BeWo choriocarcinoma cell line. Increased mitochondrial ROS resulted in a significant decrease in the transcripts which encode for proteins associated with fusion (GCM1, ERVW-1, and ERVFRD-1) resulting in a 5-fold decrease in the percentage of BeWo fusion. This outcome was associated with increased indicators of mitochondrial fragmentation, as determined by decreased expression of MFN2 and OPA1 along with an increase in a marker of mitochondrial fission (DRP1). Importantly, increased mitochondrial ROS also resulted in a 5.0-fold reduction of human placental lactogen (PL) and a 4.4-fold reduction of insulin like growth factor 2 (IGF2) transcripts; hormones which play an important role in regulating fetal growth. The pre-treatment of rotenone-exposed cells with 5 mM N-acetyl cysteine (NAC) resulted in the prevention of these ROS mediated changes in BeWo function and supports a central role for mitochondrial ROS signaling in the maintenance and function of the materno-fetal interface.

MiR-34a-5p regulates viability, invasion and apoptosis of placental trophoblastic cells via modulating CDK6 and PI3K/AKT pathway

To investigate the roles of microRNA (miR)-34a-5p and cyclin-dependent kinase (CDK) 6 in the regulation of cell viability, apoptosis and invasion of human placental trophoblastic cells and the relationship between miR-34a-5p and CDK6. We examined the expression of miR-34a-5p using RT-qPCR in cultured human trophoblast HTR-8/Svneo cells and human choriocarcinoma cell lines BeWo and JEG-3HTR-8/Svneo. HTR-8/Svneo cells transfected with a miR-34a-5p-mimic, the miR-34a-5p-inhibitor, or pcDNA-CDK6 along with the mimic group were analyzed for changes in cell proliferation using MTT assay; the apoptosis of the cells were assessed by detecting caspase 3 activity and cleaved caspase 3 protein expression, and the cell invasion was evaluated using Transwell assay. Western blotting was used to determine the protein levels of CDK6, cleaved caspase 3, and MMP-9 in the cells. The interaction between CDK6 and miR-34a-5p analyzed using a luciferase reporter assay. Transfection with the miR-34a-5p mimic significantly reduced the viability (P=0.000), suppressed the invasion (P=0.049), enhanced the cell apoptosis (P=0.018), down-regulated the expressions of MMP-9 (P=0.004) and CDK6 (P=0.014), and up-regulated caspase 3 activity (P=0.018) and cleaved caspase 3 expression (P=0.003) in cultured HTR-8/Svneo cells. CDK6 was confirmed as one of the target gene of miR-34a-5p. Transfection with pcDNA-CDK6 significantly reversed the effects of miR- 34a-5p overexpression on the cell viability (P=0.000), apoptosis (P=0.015), and invasion (P=0.046). Treatment of the cells with insulin-like growth factor 1 (IGF-1), an activator of the PI3K/AKT pathway, also significantly attenuated the effects of miR-34a- 5p overexpression on the cell viability (P=0.011), apoptosis (P=0.004), and invasion (P=0.002). miR-34a-5p promotes apoptosis and inhibits the viability and invasion of human placental trophoblastic cells by down-regulating CDK6 and inactivating the PI3K/AKT pathway.

Investigating a suitable model for the study of vitamin D mediated regulation of human placental gene expression

Transfer and metabolism of vitamin D across the human placenta is required for fetal development. However, these fundamental mechanisms are not well understood and model systems are required to help understand them. The BeWo choriocarcinoma cell line is derived from extravillous trophoblast but is used as a model for villous syncytiotrophoblast and the placental barrier. Questions have been raised about the suitability of the BeWo cell line as a model for villous trophoblast. This study compares the expression of amino acid transporters and vitamin D related genes in human term placenta with the BeWo and human embryonic kidney (HEK)293 cell lines. HEK293 cells, as transporting epithelium may be more similar to placenta. Gene expression in term placenta was much more similar to HEK293 than BeWo. This study provides further evidence that the BeWo cell line is not an appropriate model for villous trophoblast and a model that more closely represents the human placenta is now required to investigate the effects of vitamin D on the placenta ex-vivo.